DNA sequence of a gene of hydroxy-phenyl pyruvate dioxygenase and production of plants containing a gene of hydroxy-phenyl pyruvate dioxygenase and which are tolerant to certain herbicides

ABSTRACT

An isolated gene from Pseudomonas is described which expresses a hydoxy phenyl pyruvate dioxygenase. Also described are chimeric genes for introduction into plants to overexpress a hydoxy phenyl pyruvate dioxygenase and produce plants which are tolerant to herbicides.

The present invention relates to a hydroxyphenylpyruvate dioxygenase(HPPD) gene, a chimeric gene comprising this gene as coding sequence andits use to obtain plants resistant to certain herbicides.

Certain herbicides have been disclosed, such as the isoxazoles describedespecially in the French Patent Applications 95 06800 and 95 13570 andespecially isoxaflutole, a selective maize herbicide, diketonitrilessuch as those described in European Applications 0 496 630, 0 496 631,in particular 2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-CF₃-phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-2,3-Cl₂-phenyl)propane-1,3-dione,triketones described in European Applications 0 625 505 and 0 625 508,in particular sulcotrione. However, a tolerance gene to such herbicideshas not been described.

Hydroxyphenylpyruvate dioxygenase is an enzyme which catalyses theconversion reaction of para-hydroxyphenylpyruvate into homogentisate.

In addition, the amino-acid sequence of hydroxyphenylpyruvatedioxygenase from Pseudomonas sp. P.J. 874 has been described, withoutthere being a description of its role in the tolerance of the plants toherbicides (Rüetschi et al.: Eur. J. Biochem. 205, 459-466, 1992). Thisdocument does not give a description of the gene coding for thisprotein.

There have now been discovered the sequence of a gene of this type andthat such a gene can, once incorporated into plant cells, produce anover-expression or an activation of HPPD in the plants giving to thelatter an worthwhile tolerance to certain novel herbicides, such asthose of the isoxazoles family or that of the triketones.

An object of the present invention is an isolated DNA sequence of a geneof non-human origin of a non-marine bacterium, or alternatively of aplant gene, or a sequence which can hybridize with this sequence,characterized in that it expresses a hydroxyphenylpyruvate dioxygenase(HPPD).

More particularly, this sequence can be of bacterial origin, such asespecially the genus Pseudomonas or alternatively of plant origin, suchas especially of monocotyledonous or dicotyledonous plants, especiallyof Arabidopsis or of Umbelliferae, such as, for example, the carrot(Daucus carotta). It can be native or wild or possibly mutated while atthe same time fundamentally retaining a property of herbicidal toleranceagainst HPPD inhibitors, such as herbicides of the isoxazoles family orthat of the triketones.

The invention likewise relates to a process of isolating the above gene,characterized in that:

as primers, some oligonucleotides from the amino-acid sequence of anHPPD are chosen,

starting from these primers, amplification fragments are synthesized byPCR

the gene is isolated by creation and screening of a genomic bank and

the gene is cloned.

Preferably, primers from the HPPD sequence of a bacterium of the genusPseudomonas is used. Particularly preferably, they are from Poeudomonasfluorescens.

The invention also relates to the use of a gene coding for HPPD in aprocess for the transformation of plants, as a marker gene or as acoding sequence allowing tolerance to certain herbicides to be conferredon the plant. It can likewise be used, in association with other markergenes and/or coding sequences, for an agronomic property.

The coding gene can be of any origin, native or wild or possiblymutated, while at the same time fundamentally retaining a property ofherbicidal tolerance against inhibitors of HPPD, such as herbicides ofthe isoxazoles family or that of the triketones. As coding sequence,especially that described above can be used.

The transformation of plant cells can be achieved by any appropriateknown means. A series of methods consists in bombarding cells orprotoplasts with particles to which are coupled the DNA sequences.

Another series of methods consists in using, as means of transfer intothe plant, a chimeric gene inserted into a Ti plasmid of Agrobacteriumtumefaciens or Ri plasmid of Agrobacterium rhizogenes.

An object of the present invention is also a chimeric gene comprising,in the transcription direction, at least one promoter regulationsequence, a heterologous coding sequence which expresseshydroxyphenylpyruvate dioxygenase and at least one terminator orpolyadenylation regulation sequence.

The promoter regulation sequence used can be any promoter sequence of agene which is naturally expressed in plants, in particular a promoter ofbacterial, viral or plant origin, such as, for example, that of a geneof the small subunit of ribulose biscarboxylase (RuBisCo) or that of agene of α-tubulin (European Application EP No. 0 652 286), oralternatively of a plant virus gene such as, for example, that ofcauliflower mosaic virus (CAMV 19S or 35S), but any suitable promotercan be used. Preferably, recourse is made to a promoter regulationsequence which favours the overexpression of the coding sequence, suchas, for example, that comprising at least one histone promoter such asdescribed in European Application EP 0507698.

According to the invention, it is equally possible to use, inassociation with the promoter regulation sequence, other regulationsequences which are situated between the promoter and the codingsequence, such as “enhancer” transcription activators, such as, forexample, tobacco etch virus (TEV) translation activator described in theApplication WO87/07644, or of transit peptides, either single, ordouble, and in this case possibly separated by an intermediate sequence,that is to say comprising, in the transcription direction, a sequencecoding for a transit peptide of a plant gene coding for a plastidlocalization enzyme, a part of the sequence of the N-terminal maturepart of a plant gene coding for a plastid localization enzyme, then asequence coding for a second transit peptide of a plant gene coding fora plastid localization enzyme, formed by a part of the sequence of theN-terminal mature part of a plant gene coding for a plastid localizationenzyme, such as described in European Application No. 0 508 909.

It is possible to use as terminator or polyadenylation regulationsequence any corresponding sequence of bacterial origin, such as, forexample, the nos terminator of Agrobacterium tumefaciens, or even ofplant origin, such as, for example, a histone terminator such asdescribed in European Application EP No. 0 633 317.

An object of the present invention is also plant cells, ofmonocotyledonous or dicotyledonous plants, especially of crops,transformed according to one of the processes described above andcomprising in their genome an efficacious quantity of a, gene expressinghydroxyphenylpyruvate dioxygenase (HPPD). It has been observed thattransformed plants of this type have a significant tolerance to certainnovel herbicides such as the isoxazoles described especially in FrenchPatent Applications 9506800 and 95 13570 and especially of4-[4-CF₃-2-(methylsulphonyl)benzoyl]-5-cyclopropylisoxazole, andespecially isoxaflutole, a selective maize herbicide, the diketonitrilessuch as those described in European Applications 0 496 630, 0 496 631,in particular2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-CF₃-phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-2,3-Cl₂-phenyl)propane-1,3-dione,the triketones described in European Applications 0 625 505 and 0 625508, in particular sulcotrione.

Finally, an object of the invention is a method of weeding plants,especially crops, with the aid of a herbicide of this type,characterized in that this herbicide is applied to plants transformedaccording to the invention, both pre-sowing, pre-emergence andpost-emergence of the crop.

An object of the invention is also the use of the HPPD gene as a markergene in the course of the “transformation-regeneration” cycle of a plantspecies and selection on the above herbicide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the protein sequence of HPPD of Pseudomonas sp. strain P.J.874. and the theoretical nucleotide sequence of the coding part. Arrowsshow regions used for amplification.

FIG. 2 shows a restriction map of the plasmid pRP A which contains theHPPD gene of P. fluorescens A32.

FIG. 3 is a comparison of the amino acid sequence of HPPD of P.fluorescens A32 and HPPD of Pseudomonas sp. P.J. 874, showing bothconsensus sequence and differences between the sequences.

The different aspects of the invention will be better understood withthe aid of the experimental examples below.

EXAMPLE 1

Isolation of the HPPD Gene of P. fluorescens A32

Starting from the amino-acid sequence of HPPD of Pseudomonas sp. P.J.874 (published by Rüetschi U. et al., 1992, Eur. J. Biochem. 205:459-466), the sequence of different oligonucleotides is deduced in orderto amplify by PCR a part of the coding sequence of HPPD of P.fluorescens A 32 (isolated by McKellar, R. C. 1982, J. Appl. Bacteriol.,53: 305-316). An amplification fragment of the gene of this HPPD hasbeen used to screen a partial genomic bank of P. fluorescens A32 andthen to isolate the gene coding for this enzyme.

A) Preparation of genomic DNA of P. fluorescens A32

The bacteria was cultivated in 40 ml of M63 minimum medium (KH₂PO₄ 13.6g/l, (NH₄)₂SO₄ 2 g/l, MgSO₄ 0.2 g/l, FeSO₄ 0.005 g/l, pH 7 plusL-tyrosine 10 mM as the sole carbon source) at 28° C. for 48 hours.

After washing, the cells are taken up in 1 ml of lysis buffer (100 mMtris HCl, pH 8.3, 1.4 M NaCl and 10 mM EDTA) and incubated for 10minutes at 65° C. After a phenol/chloroform treatment (24:1) and achloroform treatment, the nucleic acids are precipitated by addition ofone volume of isopropanol, then taken up in 300 μl of sterile water andtreated with final 10 μg/ml RNAse. The DNA is treated afresh withphenol/chloroform, chloroform and reprecipitated by addition of 1/10 ofthe volume of 3 M sodium acetate, pH 5 and 2 volumes of ethanol. The DNAis then taken up in sterile water and determined.

B) Choice of the oligonucleotides and syntheses

Starting from the amino-acid sequence of HPPD of Pseudomonas sp. P.J.874, five oligonucleotides are chosen, two directed in the terminal NH₂direction of the protein towards the COOH terminal of the protein andthree directed in the opposite direction (see FIG. 1). The choice wasdictated by the two following rules:

a stable 3′ end of the oligonucleotide, that is to say at least twobases without ambiguity.

the smallest degeneracy possible.

The oligonucleotides chosen have the following sequences:

P1: 5′TA(C/T)GA(G/A)AA(C/T)CCIATGGG3′

P2: 5′GA(G/A)ACIGGICCIATGGA3′

P3: 5′AA(C/T)TGCATIA(G/A)(G/A)AA(C/T)TC(C/T)TC3′

P4: 5′AAIGCIAC(G/A)TG(C/T)TG(T/G/A)ATICC3′

P5: 5′GC(C/T)TT(A/G)AA(A/G)TTICC(C/T)TCIC3′

They were synthesized on a Cyclone plus DNA synthesizer of the makeMILLIPORE.

With these five oligonucleotides, the amplification fragments which mustbe obtained theoretically by PCR starting from the sequence SEQ ID No. 1have the following sizes:

with the primers P1 and P3 - - ->approximately 690 bp

with the primers P1 and P4 - - ->approximately 720 bp

with the primers P1 and P5 - - ->approximately 1000 bp

with the primers P2 and P3 - - ->approximately 390 bp

with the primers P2 and P4 - - ->approximately 420 bp

with the primers P2 and P5 - - ->approximately 700 bp

C) Amplification of a coding part of HPPD of P. fluorescens A32

The amplifications were carried out on a PERKIN ELMER 9600 PCR apparatusand with PERKIN ELMER Taq polymerase with its buffer under standardconditions, that is to say for 50 μl of reaction mixture there are dNTPat 200 μM, primers at 20 μM, 2.5 units of Taq polymerase and 2.5 μg ofDNA of P. fluorescens A32.

The amplification programme used is 5 min at 95° C. and then 35<45 sec95° C., 45 sec 49° C., 1 min 72° C.>cycles followed by 5 min at 72° C.

Under these conditions, all the amplification fragments obtained have asize compatible with the theoretical sizes given above, which is a goodindication of the specificity of the amplifications.

The amplification fragments obtained with the sets of primers P1/P4,P1/P5 and P2/P4 are ligated into pBSII SK(−) after digestion of thisplasmid by Eco RV and treatment with the terminal transferase in thepresence of ddTTP as described in HOLTON T. A. and GRAHAM M. W. 1991,N.A.R., Vol. 19, No. 5, p. 1156.

A clone of each of the three types is partially sequenced; this allowsit to be confirmed that a part of the coding region of the HPPD of P.fluorescens A32 has been well amplified in the three cases. The P1/P4fragment is retained as probe in order to screen a partial genomic bankof P. fluorescens A32 and to isolate the complete gene of the HPPD.

D) Isolation of the gene

By Southern it is shown that a 7 Kbp fragment hybridizes, afterdigestion of the DNA of P. fluorescens A32 by the restriction enzymeBamHI, with the probe HPPD P1/P4. 400 μg of DNA of P. fluorescens A32are thus digested with the restriction enzyme BamHI and the DNAfragments making up approximately 7 Kbp are purified on agarose gel.

These fragments are ligated into pBSII SK(−), the latter is digestedwith BamHI and dephosphorylated by treatment with alkaline phosphatase.After transformation in E. coli DH10b, the partial genomic bank isscreened with the probe HPPD P1/P4.

A positive clone was isolated and called pRP A. Its simplified map isgiven in FIG. 2. On this map is indicated the position of the codingpart of the HPPD gene. It is composed of 1077 nucleotides which code for358 amino acids (see SEQ ID No. 1). The HPPD of P. fluorescens A32 has agood amino-acid homology with that of Pseudomonas sp. strain P.J. 874,in fact there is 92% agreement between these two proteins (see FIG. 3).

EXAMPLE 2

Construction of Two Chimeric Genes

To confer plant tolerance to herbicides inhibiting HPPD, two chimericgenes are constructed:

The first consists in putting the coding part of the HPPD gene of P.fluorescens A32 under the control of the double histone promoter(European Patent No. 0 507 698) followed by tobacco etch virustranslational enhancer (TEV) (pRTL-GUS (Carrington and Freed, 1990; J.Virol. 64: 1590-1597)) with the terminator of the nopaline synthasegene. The HPPD will then be localized in the cytoplasm.

The second will be identical to the first, except that the optimizedtransit peptide (OTP) is intercalated between the TEV transcriptionactivator and the coding part of the HPPD (European Application EP No. 0508 909). The HPPD will then be localized in the chloroplast.

A) Construction of the vector pRPA-RD-153:

pRDA-RD-11 A derivative of pBS-II SK(−) (Stratagene catalog #212206)containing the polyadenylation site of nopaline synthase (NOS polyA)(European Application EP No. 0 652 286) is cloned between the KpnI andSalI sites. The KpmI site is transformed into a NotI site by treatmentwith T4 DNA polymerase I in the presence of 150 μM of deoxynucleotidetriphosphates and then ligation with an NotI linker (Stratagene catalog#1029). An NOS polyA cloning cassette is thus obtained.

pRPA-RD-127: A derivative of pRPA-BL-466 (European Application EP No. 0337 899) cloned in pRPA-RD-11 creating an expression cassette of the oxygene and containing the promoter of the small subunit of ribulosebiscarboxylase:

“promoter (SSU)-oxy gene-NOS polyA”

To create this plasmid, pRPA-BL-488 was digested with XbaI and HindIIIto isolate a fragment of 1.9 kbp comprising the SSU promoter and the oxygene which was ligated into the plasmid pRPA-RD-11, digested withcompatible enzymes.

pRPA-RD-132: This is a derivative of pRPA-BL-488 (European ApplicationEP No. 0 507 698) cloned into pRPA-RD-127 with creation of an expressioncassette of the oxy gene with the double histone promoter:

“double histone promoter-oxy gene-NOS polyA”

To produce this plasmid, pRPA-BL-466 is digested with HindIII, treatedwith Klenow and then redigested with NcoI. The purified fragment of 1.35kbp containing the histone double promoter H3A748 is ligated with theplasmid pRPA-RD-127 which had been digested with XbaI, treated withKlenow and redigested with NcoI.

pRPA-RD-153: This is a derivative of pRPA-RD-132 containing thetranslation activator of the tobacco etch virus (TEV). pRTL-GUS(Carrington and Freed, 1990; J. Virol. 64: 1590-1597) is digested withNcoI and EcoRI and the 150 bp fragment is ligated into pRPA-RD-132digested with the same enzymes. An expression cassette containing thepromoter:

“double histone promoter-TEV-oxy gene-NOS polyA”is thus created.

B) Construction of the vector pRPA-RD-185:

pUC19/GECA: A derivative of pUC-19 (Gibco catalog #15364-011) containingnumerous cloning sites. pUC-19 is digested with EcoRI and ligated withthe oligonucleotide linker 1:

Linker 1: AATTGGGCCA GTCAGGCCGT TTAAACCCTA GGGGGCCCG

CCCGGT CAGTCCGGCA AATTTGGGAT CCCCCGGGC TTAA

The selected clone contains an EcoRI site followed by polylinker whichcontains the following sites: EcoRI, ApaI, AvrII, PmeI, SfiI, SacI,KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII.

pRPA-RD-185: this is a derivative of pUC19/GECA containing a modifiedpolylinker. pUC19/GECA is digested with HindIII and ligated with theoligonucleotide linker 2:

Linker 2: AGCTTTTAAT TAAGGCGCGC CCTCGAGCCT GGTTCAGGG

AAATTA ATTCCGCGCG GGAGCTCGGA CCAAGTCCC TCGA

The selected clone contains a HindIII site in the centre of thepolylinker which now contains the following sites: EcoRI, ApaI, AvrII,PmeI, SfiI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI, HindIII,PacI, AscI, XhoI and EcoNI.

C) Construction of the vector pRP T:

pRP O: a derivative of pRPA-RD-153 containing an expression cassette ofHPPD, double histone promoter-TEV-HPPD gene-terminator Nos. To producepRP O, pRPA-RD153 is digested with HindIII, treated with Klenow and thenredigested with NcoI to remove the oxy gene and replace it by the HPPDgene coming from the pRP A plasmid by BstEII digestion, Klenow treatmentand redigestion with NcoI.

pRP R: to obtain the plasmid, pRP O was digested with PvuII and SacI,the chimeric gene was purified and then ligated into pRPA-RD-185 and thelatter was digested with PvuII and SacI.

pRP T: was obtained by ligation of the chimeric gene-coming from pRP Rafter digestion with SacI and HindIII in the plasmid pRPA-BL 150 alpha2digested with the same enzymes (European Application EP No. 0 508 909).

The chimeric gene of the pRP T vector thus has the following structure:

Double histone TEV Coding region nos promoter of HPPD terminator

D) Construction of the pRP V vector

pRP P: this is a derivative of pRPA-RD-7 (European Application EP No. 0652 286) containing the optimized transit peptide followed by the HPPDgene. It was obtained by ligation of the coding part of HPPD coming frompRP A by BetEII and NcoI digestion, Klenow treatment and from theplasmid pRPA-RD-7, the latter digested with SphI and AccI and treatedwith DNAse polymerase T4.

pRP Q: a derivative of pRPA-RD-153 containing an expression cassette ofHPPD, double histone promoter-TEV-OTP-HPPD gene-Nos terminator. Toconstruct it, the plasmid pRPA-RD-153 is digested with SalI, treatedwith Klenow and then redigested with NcoI to remove the oxy gene andreplace it by the HPPD gene released from the pRP P plasmid by BstEIIdigestion, Klenow treatment and redigestion with NcoI.

pRP S: to obtain it, the plasmid pRP Q was digested with PvuII and SacIto release the chimeric gene, which was ligated into pRPA-RD-185, thelatter digested with PvuII and SacI.

pRP V: it was obtained by ligation of the chimeric gene released frompRP S, after digestion with SacI and HindIII, into the plasmid pRPA-BL150 alpha2 (European Application EP No. 0 508 909).

The chimeric gene of the pRP Q vector thus has the following structure:

Double histone TEV OTP Coding region nos promoter of HPPD terminator

EXAMPLE 3

Transformation of the Industrial Tobacco PBD6

In order to determine the efficacy of these two chimeric genes, thesewere transferred to industrial tobacco PBD6 according to thetransformation and regeneration procedures already described in EuropeanApplication EP No. 0 508 909.

1) Transformation

The vector is introduced into the non-oncogenic strain of AgrobacteriumEHA 101 (Hood et al., 1987) which carries the cosmid pTVK 291 (Komari etal., 1986). The transformation technique is based on the procedure ofHorsh R. et al. (1985), Science, 227, 1229-1231.

2) Regeneration

The regeneration of the tobacco PBD6 (origin SEITA France) from foliarexplants is carried out on a Murashige and Skoog (MS) base mediumcomprising 30 g/l of sucrose as well as 200 μg/ml of kanamycin. Thefoliar explants are selected on plants in the greenhouse or in vitro andtransformed according to the foliar discs technique (Science 1985, Vol.227, p. 1229-1231) in three successive steps: the first comprises theinduction of shoots on an MS medium to which is added 30 g/l of sucrosecontaining 0.05 mg/l of naphthylacetic acid (NAA) and 2 mg/l ofbenzylaminopurine (BAP) for 15 days. The shoots formed during this stepare then developed by culture on an MS medium to which is added 30 g/lof sucrose, but not containing any hormone, for 10 days. Developedshoots are then selected and cultured on an MS rooting medium of halfsalts, vitamins and sugars content and not containing any hormone. Atthe end of approximately 15 days, the rooted shoots are placed in earth.

EXAMPLE 4

Measurement of the Tolerance of the Tobacco to4-[4-CF₃-2-(methylsulphonyl)benzoyl]-5-cyclopropylisoxazole:Post-Emergence Treatment

On leaving in-vitro culture, the transformed tobacco plantlets wereacclimatized in a greenhouse (60% relative humidity; temperature: 20° C.during the night and 23° C. during the day) for five weeks and thentreated with4-[4-CF₃-2-(methylsulphonyl)benzoyl]-5-cyclopropylisoxazole.

The control tobacco, non-transformed and treated with4-[4-CF₃-2-(methylsulphonyl)benzoyl]-5-cyclopropylisoxazole in dosesranging from 50 to 400 g/ha, develops chloroses in approximately 72hours, which intensify to develop into very pronounced necroses in aweek (covering approximately 80% of the terminal leaves).

After transformation, this same tobacco, which overexpresses the HPPD ofP. fluoreacens, is very well protected against treatment with4-[4-CF₃-2-(methylsulphonyl)benzoyl]-5-cyclopropylisoxazole at a dose of400 g/ha.

If the overexpressed enzyme is in the cytoplasm, that is to say if thetransformation was carried out with the gene carried by the vector pRPT, then the plant shows very slight chloroses which are all localized onthe intermediate leaves.

If the overexpressed enzyme is in the chloroplast, that is to say if thetransformation was carried out with the gene carried by the vector pRPV, then the plant is perfectly protected and does not show any symptoms.

EXAMPLE 5

Measurement of the Tolerance of the Tobacco to 4- [4-CF₃-2-(methylsulphonyl)benzoyl]-5-cyclopropylisoxazole: Pre-EmergenceTreatment

a) in vitro test

Tobacco seeds harvested from plants from the“transformation-regeneration” cycle and resistant to isoxaflutole foliartreatment are used at a dose of 400 g/h described in Examples 1 to 3.

These seeds were sown in boxes containing plant agar at 10 g/l andisoxaflutole at different concentrations ranging from 0 to 1 mg/l.Germination was then carried out at 25° C. with a photoperiod of 12hours of light/12 hours of darkness.

According to this protocol, wild tobacco seeds were germinated as wellas seeds of the two types of transgenic tobacco, that is to say CYtobaccos, with localization of the HPPD in the cytoplasm, and the COtobaccos with localization of the HPPD in chloroplast.

Resistance measurements are carried out visually between 2 and 3 weeksafter sowing.

The results are recorded in the table below.

isoxaflutole concentra- tion Wild tobacco CY tobacco CO tobacco 0 mg/l100% of the 100% of the 100% of the seeds ger- seeds seeds minategerminate germinate without without without symptoms° symptoms° symptoms0.05 mg/l 20% of the 75% of the 75% of the seeds seeds seeds germinategerminate* germinate* and show without without symptoms° symptoms°symptoms° 0.1 mg/l no 75% of the 75% of the germination seeds seedsgerminate* germinate* without without symptoms° symptoms° 0.5 mg/l no75% of the 75% of the germination seeds seeds germinate* germinate*without without symptoms° symptoms° 1 mg/l no 75% of the 75% of thegermination seeds seeds germinate* germinate* with slight withoutsymptoms° symptoms° °the symptoms which the plantlets show in the courseof germination are more or less significant deformations of thecotyledons and above all a bleaching of the tissues which are normallyphotosynthetic and thus green. *75% of the seeds germinate because seedsfrom the self-fertilization of single-locus plants coming from the“transformation-regeneration” cycle and thus only carrying the tolerancegene on a chromosome were sown. Working in the same way with thefollowing products, Product No. 51 of American Patent 4 780 127, thesame results are obtained at a concentration of 0 mg/l and 0.1 mg/l onwild tobacco and CO tobacco.

b) Greenhouse Test

Measurement is carried out as in Example 4, apart from the treatmentbeing carried out pre-emergence, 24 hours before sowing. Wild sowing iscarried out normally. Under these conditions, it is observed that, forthe non-treated control sowings, there is no germination for any dose ofherbicide at least equal to 10 g/ha. On the contrary, the CY tobaccos donot show any symptoms, such as defined in paragraph a), up to andincluding 100 g/ha. Similarly, the CO tobaccos do not show any symptoms,such as defined in paragraph a), up to and including 200 g/ha.

These results show clearly that the HPPD gene of P. fluorescens confersa tolerance to the tobacco against pre-emergence treatments withisoxaflutole. This tolerance is better if the protein is localized inthe chloroplast in place of the cytoplasm.

EXAMPLE 6

With the aim of studying whether the HPPD gene of Pseudomonasfluorescens can be used as a marker gene in the course of the“transformation-regeneration” cycle of a plant species, tobacco wastransformed with the HPPD gene and transformed plants were obtainedafter selection on isoxaflutole.

Material and Methods and Results

The chimeric gene pRP V described below is transferred into industrialtobacco PBD6 according to the transformation and regeneration proceduresalready described in European Application EP No. 0 508 909.

The chimeric gene of the vector pRP V has the following structure:

Double histone TEV OTP Coding region nos promoter of HPPD terminator

1) Transformation

The vector is introduced into the Agrobacterium EHA 101 non-oncogenicstrain (Hood et al., 1987) which carries the cosmid pTVK 291 (Komari etal., 1986). The transformation technique is based on the procedure ofHorsh et al. (1985).

2) Regeneration

The regeneration of the tobacco PBD6 (origin SEITA France) from foliarexplants is carried out on a Murashige and Skoog (MS) base mediumcomprising 30 g/l of sucrose as well as 350 mg/l of cefotaxime and 1mg/l of isoxaflutol. The foliar explants are selected on plants in agreenhouse or in vitro and transformed according to the foliar discstechnique (Science 1985, Vol. 227, p. 1229-1231) in three successivesteps: the first comprises the induction of shoots on an MS medium towhich is added 30 g/l of sucrose containing 0.05 mg/l of naphthylaceticacid (NAA) and 2 mg/l of benzylaminopurine (BAP) for 15 days and 1 mg/lof isoxaflutole. The green shoots formed in the course of this step arethen developed by culture on an MS medium to which is added 30 g/l ofsucrose and 1 mg/l of isoxaflutole, but not containing hormone, for 10days. Developed shoots are then selected and are cultured on an MSrooting medium of half salts, vitamins and sugars content and 1 mg/l ofisoxaflutole and not containing any hormone. At the end of approximately15 days, the rooted shoots are placed in earth.

All the plantlets obtained according to this protocol are analysed byPCR with specific primers of the HPPD of P. fluorescens. This PCRanalysis has enabled it to be confirmed that all the plantlets thusobtained have well integrated the HPPD gene.

In conclusion, this assay confirms that the HPPD gene can be used asmarker gene and that, associated with this gene, isoxaflutole can be agood selection agent.

EXAMPLES 7 AND 8

Isolation of the HPPD Gene of Arabidopsis Thaliana and of the HPPD Geneof Carrot (Daucus Carotta)

a) Construction of CDNA Banks mRNAs extracted from young plantlets ofArabidopsis thaliana and mRNAs extracted from carrot cells in cultureserved to construct two cDNA banks in the vector Uni Zap™ XR marketed bythe company Stratagen, following the protocol recommended by thiscompany.

b) Screening of the cDNA Banks These two banks were screened with theaid of a probe corresponding to a cDNA of Arabidopsis thaliana ofpartial length, obtained via the Arabidopsis Biological Resource Center(Ohio, USA) and indexed: EST clone No. 91B13T7. This clone is formed ofapproximately 500 base pairs of which only 228 have been sequenced bythe MSU-DOE Plant Research Laboratory in the context of randomsequencing of cDNA of Arabidopsis thaliana. We completely sequenced the500 base pairs before using this clone to screen our cDNA banks ofArabidopsis thaliana and of carrot with the aid of the classicaltechnique of hybridization of lysis plaques (reference ?).

c) A cDNA of Arabidopsis thaliana (SEQ ID No. 2) corresponding to 1338base pairs was obtained. This cDNA has a translation initiation startcodon in position 25 and a translation end codon in position 1336. ThiscDNA is thus complete and codes for a protein of 445 amino acids.

d) A cDNA of carrot (Daucus carotta) (SEQ ID No. 3) corresponding to1329 base pairs was obtained. This cDNA has a translation initiationstart codon in position 1 and a translation finish codon in position1329. This cDNA is thus complete and codes for a protein of 442 aminoacids.

The sequences illustrated are the following:

SEQ ID No. 1 Sequence of the HPPD gene of Pseudomonas fluorescens A32

SEQ ID No. 2

cDNA sequence of HPPD of Arabidopsis thaliana

SEQ ID No. 3

cDNA sequence of HPPD of Daucus carotta

FIG. 1 represents the protein sequence of the HPPD of Pseudomonas sp.strain P.J 874 and the theoretical nucleotide sequence of thecorresponding coding part; the five oligomucleotides chosen to carry outthe amplification of a part of.this coding region are symbolized by thefive arrows.

FIG. 2 represents the mapping of the plasmid with the genomic DNAfragment of 7 kb comprising the gene of the HPPD of P. fluorescens A32.

FIG. 3 gives the comparison of the amino-acid sequences of the HPPD of Pfluorescens A32 and of the HPPD of Pseudomonas sp. strain P.J.874 (onlythe divergant amino acids, between the two sequences are indicated) aswell as the concensus sequence.

3 1 1077 DNA Pseudomonas 1 atggcagatc tatacgaaaa cccaatgggc ctgatgggctttgaattcat cgaattcgcg 60 tcgccgacgc cgggtaccct ggagccgatc ttcgagatcatgggcttcac caaagtcgcg 120 acccaccgtt ccaagaacgt gcacctgtac cgccagggcgagatcaacct gatcctcaac 180 aacgagccca acagcatcgc ctcctacttt gcggccgaacacggcccgtc ggtgtgcggc 240 atggcgttcc gcgtgaagga ctcgcaaaag gcctacaaccgcgccctgga actcggcgcc 300 cagccgatcc atattgacac cgggccgatg gaattgaacctgccggcgat caagggcatc 360 ggcggcgcgc cgttgtacct gatcgaccgt ttcggcgaaggcagctcgat ctacgacatc 420 gacttcgtgt acctcgaagg tgtggagcgc aatccggtcggtgcaggtct caaagtcatc 480 gaccacctga cccacaacgt ctatcgcggc cgcatggtctactgggccaa cttctacgag 540 aaattgttca acttccgtga agcgcgttac ttcgatatcaagggcgagta caccggcctg 600 acttccaagg ccatgagtgc gccggacggc atgatccgcatcccgctgaa cgaagagtcg 660 tccaagggcg cggggcagat cgaagagttc ctgatgcagttcaacggcga aggcatccag 720 cacgtggcgt tcctcaccga cgacctggtc aagacctgggacgcgttgaa gaaaatcggc 780 atgcgcttca tgaccgcgcc gccagacact tattacgaaatgctcgaagg ccgcctgcct 840 gaccacggcg agccggtgga tcaactgcag gcacgcggtatcctgctgga cggatcttcc 900 gtggaaggcg acaaacgcct gctgctgcag atcttctcggaaaccctgat gggcccggtg 960 ttcttcgaat tcatccagcg caagggcgac gatgggtttggcgagggcaa cttcaaggcg 1020 ctgttcgagt ccatcgaacg tgaccaggtg cgtcgtggtgtattgaccgc cgattaa 1077 2 1338 DNA Arabidopsis 2 atgggccacc aaaacgccgccgtttcagag aatcaaaacc atgatgacgg cgctgcgtcg 60 tcgccgggat tcaagctcgtcggattttcc aagttcgtaa gaaagaatcc aaagtctgat 120 aaattcaagg ttaagcgcttccatcacatc gagttctggt gcggcgacgc aaccaacgtc 180 gctcgtcgct tctcctggggtctggggatg agattctccg ccaaatccga tctttccacc 240 ggaaacatgg ttcacgcctcttacctactc acctccggtg acctccgatt ccttttcact 300 gctccttact ctccgtctctctccgccgga gagattaaac cgacaaccac agcttctatc 360 ccaagtttcg atcacggctcttgtcgttcc ttcttctctt cacatggtct cggtgttaga 420 gccgttgcga ttgaagtagaagacgcagag tcagctttct ccatcagtgt agctaatggc 480 gctattcctt cgtcgcctcctatcgtcctc aatgaagcag ttacgatcgc tgaggttaaa 540 ctatacggcg atgttgttctccgatatgtt agttacaaag cagaagatac cgaaaaatcc 600 gaattcttgc cagggttcgagcgtgtagag gatgcgtcgt cgttcccatt ggattatggt 660 atccggcggc ttgaccacgccgtgggaaac gttcctgagc ttggtccggc tttaacttat 720 gtagcggggt tcactggttttcaccaattc gcagagttca cagcagacga cgttggaacc 780 gccgagagcg gtttaaattcagcggtcctg gctagcaatg atgaaatggt tcttctaccg 840 attaacgagc cagtgcacggaacaaagagg aagagtcaga ttcagacgta tttggaacat 900 aacgaaggcg cagggctacaacatctggct ctgatgagtg aagacatatt caggaccctg 960 agagagatga ggaagaggagcagtattgga ggattcgact tcatgccttc tcctccgcct 1020 acttactacc agaatctcaagaaacgggtc ggcgacgtgc tcagcgatga tcagatcaag 1080 gagtgtgagg aattagggattcttgtagac agagatgatc aagggacgtt gcttcaaatc 1140 ttcacaaaac cactaggtgacaggccgacg atatttatag agataatcca gagagtagga 1200 tgcatgatga aagatgaggaagggaaggct taccagagtg gaggatgtgg tggttttggc 1260 aaaggcaatt tctctgagctcttcaagtcc attgaagaat acgaaaagac tcttgaagcc 1320 aaacagttag tgggatga1338 3 1329 DNA Daucas Carota 3 atggggaaaa aacaatcgga agctgaaattctctcaagca attcatcaaa cacctctcct 60 gcaacattca agctggtcgg tttcaacaacttcgtccgcg ccaaccccaa gtccgatcac 120 ttcgccgtga agcggttcca ccacattgagttctggtgcg gcgacgccac caacacgtcg 180 cggcggttct cgtggggcct cggcatgcctttggtggcga aatcggatct ctctactggc 240 aactctgttc acgcttctta tcttgttcgctcggcgaatc tcagtttcgt cttcaccgct 300 ccttactctc cgtccacgac cacttcctctggttcagctg ccatcccgtc tttctcggcc 360 tcgggttttc actcttttgc ggccaagcacggccttgctg ttcgggctat tgctcttgaa 420 gttgctgacg tggctgctgc gtttgaggccagtgttgcgc gtggggccag gccggcgtcg 480 gctcctgttg aactgggcga ccaggcgtggttggcggagg tggagttgta cggagatgtg 540 gtcttgaggt ttgttagttt tgggagggaggagggtttgt ttttgcctgg attcgaggcg 600 gtggagggga tggcgtcgtt tccggatttggattatggaa ttagaagact tgatcatgcg 660 gtggggaatg ttaccgagtt ggggcctgtggtggagtata ttaaagggtt tacggggttt 720 catgaatttg cggagtttac agcggaggatgtggggactt tggagagtgg gttgaattcg 780 gtggtgttgg cgaataacga ggagatggttctgttgccct tgaatgagcc tgtgtatggg 840 accaagagga agagtcagat acagacttacttggagcaca atgaaggggc tggagtgcag 900 catttggctt tagtgagtga ggatatttttaggactttga gggagatgag gaagaggagt 960 tgcctcggtg gttttgagtt tatgccttcgccaccgccta cgtattacaa gaatttgaag 1020 aatagggtcg gggatgtgtt gagtgatgaacagatcaagg agtgtgaaga tttggggatt 1080 ttggtggata gggatgatca gggtacattgcttcaaatct ttaccaagcc tgtaggtgac 1140 aggcctacct tattcataga gatcattcagagggtagggt gcatgctcaa ggatgatgca 1200 gggcagatgt accagaaggg cgggtgcggaggatttggga aggggaactt ctcagagctg 1260 ttcaagtcca tcgaagaata tgaaaaaacacttgaagcta aacaaatcac tggatctgct 1320 gctgcatga 1329

What is claimed is:
 1. An isolated gene which expresses a hydroxyphenylpyruvate dioxygenase (HPPD) from Pseudomonas.
 2. Sequence according to claim 1, characterized in that it is from Pseudomonas fluorescens.
 3. A chimeric gene comprising, in the direction of transcription: at least one promoter regulation sequence from a gene expressing itself naturally in plants, a heterologous coding sequence under control of the promoter regulation sequence, and at least one polyadenylation sequence, characterized in that the coding sequence expresses hydroxyphenylpyruvate diogenase (HPPD).
 4. Chimeric gene according to claim 3, characterized in that the promoter regulation sequence comprises at least one histone promoter.
 5. Chimeric gene according to claim 3, characterized in that it comprises, between the promoter regulation sequence and the coding sequence, a transit peptide.
 6. Chimeric gene according to claim 3, characterized in that it comprises, between the promoter regulation sequence and the coding sequence and, in the transcription direction, a sequence coding for a transit peptide of a plant gene coding for a plastid localization enzyme, a part sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme, then a sequence coding for a second transit peptide of a plant gene coding for a plastid localization enzyme.
 7. Chimeric gene according to claim 3, characterized in that it comprises, between the promoter regulation sequence and the coding sequence, a sequence of a transcription activator (enhancer).
 8. A vector comprising a chimeric gene according to claim
 3. 9. A plant which contains the chimeric gene as claimed in claim
 3. 10. A method of selective herbicidal treatment of plants, which comprises applying an HPPD-inhibiting herbicide to the plant as claimed in claim
 9. 11. The method of claim 10 wherein the herbicide is an isoxazole.
 12. The method of claim 11, wherein the isoxazole is4-[4-CF₃-2-methylsulfonylbenzoyl]-5-cyclopropyl isoxazole.
 13. The method of claim 10 wherein the herbicide is a diketonitrile.
 14. The method of claim 10 wherein the herbicide is a triketone.
 15. The method of claim 10 wherein the herbicide is a sulcotrione. 